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Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

Background: OXPHOS mediates resistance to BRAF and MEK inhibitors in melanoma. Recently, we showed that MBMs have higher expression of OXPHOS genes than patient-matched extracranial metastases (ECMs). The induction of, and dependence upon, OXPHOS in MBMs was confirmed in mouse xenograft models. Notably, our studies demonstrated that OXPHOS varies among MBMs. In order to improve our understanding of the High-OXPHOS phenotype, we report here the molecular, immune, metabolic, and radiomic features that correlate with OXPHOS levels in MBMs. Methods: We applied an OXPHOS gene signature to RNA-sequencing (RNA-seq) data from surgically resected MBMs (n=88). Clustering by these genes identified MBMs with significant enrichment (High-OXPHOS; n=21) and depletion (Low-OXPHOS; n=25) of OXPHOS genes. The EdgeR/limma/voom pipeline was used to perform differential gene expression (DGE) analysis between High- and Low-OXPHOS MBMs. Pathway analyses were performed via Ensemble of Gene Set Enrichment Analyses (EGSEA). The ESTIMATE and MCP-Counter R packages were used to assess immune infiltrates from voom-transformed counts. Quantitative analysis of P-S6 and P-PRAS40 was performed by immunohistochemistry (IHC). Tumor phenotypes were segmented on conventional magnetic resonance (MR) images, and radiomic features were extracted using an in-house radiome-sequencing pipeline. Radiomic features were utilized to predict High- vs. Low-OXPHOS status. To evaluate the robustness of the estimates, Leave One Out cross-validation (LOOCV) was conducted on the patient set. Direct metabolite analysis was performed via liquid chromatography mass spectrometry (LC-MS) of Low-OXPHOS A375 and High-OXPHOS, MEKi-resistant A375-R1 brain xenografts. Results: RNA-seq analysis identified that High-OXPHOS MBMs are characterized by significant enrichment of PGC1α (FDR q-val=0.0002) and mTOR (FDR q-val=0.001) signaling and significant depletion of ImmuneScores, T cells, cytotoxic lymphocytes, B lineage cells, and NK cells (p Conclusions: Significant differences in mTOR (enriched) and immune (depleted) signaling were identified in High-OXPHOS clinical MBMs, and enrichment of glutamine signaling was observed in High-OXPHOS MBM xenografts. Further, our results demonstrated that changes in MR radiomic features can noninvasively discriminate between High- and Low-OXPHOS MBMs. Additional studies are ongoing to evaluate the clinical, prognostic, and predictive values of OXPHOS status in melanoma. Citation Format: Grant M. Fischer, Aikaterini Kotrotsou, Sara Ahmed, Pilar O’Neal, Iman Sahnoune, Y.N. Vashisht Gopal, Courtney Hudgens, Barbara Knighton, Rivka Colen, Michael T. Tetzlaff, Michael A. Davies. Molecular, immunologic, metabolic, and radiomic associations of oxidative phosphorylation (OXPHOS) in melanoma brain metastases (MBMs) [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A05.

Background: The gut microbiome is increasingly being recognized as a strong modulator of anti-PD1 based cancer immunotherapy. Compelling evidence demonstrates differential bacterial enrichment and diversity in responders (R) versus non-responders (NR), mediated by profound influences on systemic and anti-tumor immune infiltrates. However, this has not been studied in the setting of treatment with combined immune checkpoint blockade (CICB), which is associated with superior response rates, but higher rates of potentially debilitating toxicities. Methods: We assembled a cohort of patients with metastatic melanoma receiving CICB (n=54). All patients were classified as R (n=31, CR + PR) or NR (n=23, SD + PD) based on RECIST v1.1, and as having grade 3 or higher (T; n=29), or less than grade 3 (NT; n=25) immune related adverse event(s) by NCI CTCAE 4.0 criteria. Baseline stool samples were characterized by 16S rRNA sequencing. Correlative analyses of peripheral immune cell populations by flow cytometry (n=12) and circulating T cell repertoire by TCR-sequencing (n=12) were done on matched pre-treatment blood samples. Results: The overall gut microbial landscape in these patients was varied with high abundance of Bacteroidales and Clostridiales. Ordination of beta-diversity distances revealed a lack of clustering by subtype of primary tumor (uveal, mucosal, cutaneous) consistent with no significant effect of the tumor histology. While no apparent response or toxicity associations were evident based on diversity, notable compositional differences were appreciated. Comparison of relative abundances by LEfSe (LDA>2, p Conclusion: These findings build on our prior work and support the notion of a close link between the gut microbiome and therapeutic outcomes to checkpoint blockade therapy. Extensive studies are underway in both matched human biospecimens and in pre-clinical models to further understand mechanisms of interactions with immune markers, and to establish causality. Taken together, these data support a critical role for the gut microbiome as both a predictive tool and therapeutic target. Citation Format: Vancheswaran Gopalakrishnan, Miles Andrews, Wei-Shen Chen, Christine Spencer, Luis Vence, Alexandre Reuben, Zachary A. Cooper, Peter A. Prieto, Michael T. Tetzlaff, MA Abdul Wadud Khan, Alexander Lazar, Courtney W. Hudgens, Lauren E. Haydu, Hussein A. Tawbi, Patrick Hwu, Wen-Jen Hwu, Rodabe N. Amaria, Elizabeth M. Burton, Scottt E. Woodman, Adi Diab, Sapna P. Patel, Isabella C. Glitza, Jianhua Zhang, Joseph Petrosino, Robert R. Jenq, Michael A. Davies, Jeffrey E. Gershenwald, Padmanee Sharma, James P. Allison, Andrew Futreal, Jennifer A. Wargo. Therapeutic efficacy and tolerability of combined immune checkpoint blockade in metastatic melanoma patients is influenced by the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1493.

Background: While treatment with immune checkpoint blockade (ICB) has markedly improved outcomes in advanced melanoma patients and other malignancies, predicting response remains a challenge. Biomarkers including tumor mutational burden (TMB), T-cell infiltration, and PD-L1 expression, have been identifiedbut remain inadequate. Other components of innate and adaptive immunity, including B-cells and tertiary lymphoid structures (TLS), have been implicated in the response to other cancer therapies, andpreclinical data suggests B cells may contribute in the response to immunotherapy. Here, we use targeted protein expression profiling-via NanoString digital spatial profiling (DSP) technology (research use only)-to demonstrate a role for B cells in the response to ICB in patients with high-risk resectable melanoma; furthermore, we characterize the B cell subsets that enable this function. Methods: We conducted a phase 2 clinical trial of neoadjuvant ICB therapy in patients with high-risk resectable melanoma (PD-1 blockade monotherapy or combined CTLA-4/PD-1 blockade) (NCT02519322). Longitudinal tumor samples were taken during therapy. Formalin-fixed paraffin embedded tissue sections from tumor samples (n=10 responders [R], n=10 non-responders [NR]) were analyzed by NanoString DSP technology and stained with a cocktail of S100B, CD45, CD19 and a 40-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. Regions of interest (ROI) were delineated using immunofluorescence followed by UV excitation of the defined ROIs, releasing the DNA barcodes for downstream quantitation on the NanoString nCounter®platform. Utilizing masking strategies, we define the unique expression pattern within discrete subsets of immune cells. These same tumors have concurrently been analyzed by RNAseq, immunohistochemistry, CYTOF, and single-cell RNAseq. Results: Biomarker counts are highly concordant across samples from the same patient’s tumor. High concordance between DSP and quantitative fluorescence is seen as a validation for the DSP method. We identify B cells as components of TLS; the B cells are closely integrated with CD4 and CD8 T cells and follicular dendritic cells. There are significantly more TLS identified in R compared to NR patients. Utilizing CYTOF, we concurrently identify specific subsets of B cells present within the TLS associated with response. We characterize the spatial relationship of these same B cell subsets with other components of the TLS and define the protein expression patterns of these cells. Conclusion: NanoString DSP data complement our deep molecular and immune profiling of tumors from melanoma patients treated with ICB; together, they provide a novel predictive role for B-cells and TLS in the response to ICB and, importantly, provide mechanistic insight into their potential contribution in the response to cancer therapy. Citation Format: Beth A. Helmink, Sangeetha Reddy, Jorge Blando, Yan Liang, Sarah Warren, Vancheswaran Gopalakrishnan, Hussein A. Tawbi, Rodabe N. Amaria, Michael Davies, Jeffrey E. Gershenwald, Elizabeth Burton, Rafet Basar, Alexander J. Lazar, Courtney W. Hudgens, Katy Rezvani, James P. Allison, Padmanee Sharma, Joseph M. Beechem, Jennifer A. Wargo, Michael T. Tetzlaff. NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 499.

Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy. Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors. Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC. Citation Format: Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1719.

BACKGROUND: MBMs are a common and lethal complication of advanced melanoma. An improved understanding of the molecular features of MBMs could facilitate the development of more effective treatments for patients (pts). Further, identification of differences between MBMs and ECMs would indicate the need for organ-specific treatment strategies. METHODS: Transcriptomic capture and Illumina RNA-sequencing (RNA-seq) were performed on (i) surgically resected formalin-fixed, paraffin-embedded (FFPE) MBMs (98 tumors from 84 pts) and (ii) surgically-resected ECMs from the same pts (54 from 38 pts). The EdgeR/limma/voom pipeline was used to perform differential gene expression (DGE) analyses. Pathway analyses were performed via Ensemble Gene Set Enrichment Analysis (EGSEA). The ESTIMATE and MCPCounter R packages were used to assess immune infiltrates from voom-transformed counts. Illumina whole exome sequencing (WES) was performed on 27 matched pairs of MBMs and ECMs from 17 pts. MuTect algorithm was utilized to assess non-synonymous, stop-gain, and stop-loss mutations. DNA methylation profiling was performed on 16 pt-matched pairs of MBMs and ECMs by the Illumina MethylationEPIC BeadChip platform; data was analyzed via the ChAMP pipeline. Quantitative analysis of CD3 and CD8 immune markers was performed by immunohistochemistry (IHC) on 31 MBMs and 49 pt-matched ECMs. RESULTS: Overall patterns of mRNA expression, mutational burden, and gene methylation were largely similar between pt-matched pairs of MBMs and ECMs, as unsupervised hierarchical clustering was driven primarily by patient identification. However, EGSEA of RNA-seq data identified significant (FDR q-value < 0.10) differences in immune networks (decreased in MBMs) and neuronal differentiation factors (increased in MBMs) in the pt-matched tumors. MCPCounter analysis revealed significant (p < 0.05) depletion of all classes of immune cells except neutrophils in MBMs compared to ECMs. IHC confirmed decreased TCD3 (p = 0.005) and TCD8 (p = 0.013) infiltrating cells in MBMs. ESTIMATE ImmuneScores among MBMs inversely correlated with expression of VSIG4, a relative of the B7 family believed to inhibit T-cell proliferation, and were significantly (p = 0.0088) increased in MBMs previously treated with radiation (XRT). Increased ImmuneScores also associated with significantly (p = 0.00002) improved overall survival (OS) from surgery for MBM. Significant correlations in mean promoter region methylation changes and RNA-seq log fold-changes were identified in 5/10 (50%) of the neuronal factors overexpressed in MBMs, suggesting an epigenetic mechanism for their differential expression. CONCLUSIONS: Significant differences in immune and neuronal gene networks were detected in MBMs compared to patient-matched ECMs, and expression of immune genes in MBMs positively correlated with previous XRT and OS. Citation Format: Grant M. Fischer, Ali Jalali, Aron Joon, Michael T. Tetzlaff, Alexander J. Lazar, Fernando Carapeto, Mariana P. Macedo, Courtney W. Hudgens, Jennifer L. McQuade, Khalida Wani, Brandy Conner, Bhavana Singh, Michael A. Davies. Comprehensive molecular profiling of melanoma brain metastases (MBMs) and patient (pt)-matched extracranial metastases (ECMs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3392.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin tumor. MCC is immunogenic and highly responsive to immune checkpoint inhibitors. On rare occasions, MCC undergoes spontaneous regression of both local and metastatic disease in the absence of treatment. With at least 45 cases reported in the literature, this phenomenon has been described in 1.7-3% of MCCs but its mechanism is poorly understood. There has been speculation that biopsy-induced antigen shedding and subsequent recruitment of a host immune response drives spontaneous tumor regression. Here, we demonstrate activation of an adaptive immune response in a case of spontaneous MCC regression. A 65 year-old man presented with a rapidly-growing 3.5 cm skin tumor confirmed by histopathologic and immunohistochemical analyses to be Merkel cell polyomavirus-positive MCC. Eight days following the diagnostic shave biopsy, the tumor had clinically regressed. Histological analysis of a wide local excision specimen obtained 45 days after the initial biopsy revealed small, residual tumor nests and a surrounding lymphohistiocytic inflammatory infiltrate consistent with a regressing tumor. To evaluate the immune response at baseline and in the regressing tumor, we performed T cell receptor (TCR) sequencing as a metric of antigen-specific lymphocyte activation, and quantitative immunohistochemical analyses to phenotype the immune response. Both the baseline and regressing tumor contained intratumoral inflammatory infiltrates which were sparse relative to peritumoral infiltrates. There were notable reductions in CD4+ T cells and CD68+ macrophages in the regressing tumor relative to the initial biopsy. TCR clonality was increased in the regressing tumor relative to the baseline lesion, including both increased representation of the most dominant T cell clone from the baseline biopsy as well as emergence of new high-frequency clones. Together, these results suggest that spontaneous MCC regression is driven by expansion of novel and pre-existing adaptive cellular immune responses. Consistent with this, we observed analogous results with immunohistochemical staining and TCR sequencing performed in an MCC tumor regressing during treatment with the PD-L1 inhibitor, avelumab. Although further studies are needed to determine how biopsy unmasks immune responses and to elucidate target antigens of the T cell clones driving spontaneous MCC regression, our observations demonstrate that comparable immune activation underlies both spontaneous tumor regression and MCC response to immunotherapy. Citation Format: Mairead Baker, John W. Roman, Alexandre Reuben, Natasha Hill, Courtney W. Hudgens, Michael Tetzlaff, Nicholas F. Logemann, Isaac Brownell. Spontaneous regression of Merkel cell carcinoma is driven by adaptive immune activation and clonal T cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4676.

Prognosis is favorable in patients with primary localized melanoma but poor in patients with metastatic disease. With more than 76,000 cases expected to be diagnosed in 2016, more precise prognostic technologies and new therapies are needed. Although targeted treatment regimens have been approved in recent years, resistance has emerged in large part due to adaptive response mechanisms and intratumoral heterogeneity. Analysis of tumor samples across multiple molecular platforms will help elucidate the complexities within and across tumors which may underlie response to therapy as well as assist in identifying predictive biomarkers; however, these approaches require significant amounts of sample, time, and resources. In order to integrate the strengths of analyzing different molecular analytes, we have modularized Nanostring Technologies' molecular barcoding technology to permit simultaneous digital measurement of cancer-associated DNA mutation variants, mRNA expression, and protein expression in one assay from the same sample (3D Biology). Novel nucleotide variant probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Gene expression is measured via unique digital barcoding technology to measure mRNA transcripts, and protein expression and activity (via phosphorylation) is measured by DNA-labeled antibodies. The multi-omic workflow requires only two 5-10 micron sections of FFPE tissue, whereby DNA and RNA are extracted from one section and multiplex digital protein profiling is conducted on the second. As proof of concept demonstrating the utility of this 3D Biology platform, we have simultaneously analyzed DNA variants, RNA expression, and protein expression using NanoString’s nCounter Vantage 3D™ Solid Tumor Panel on 12 FFPE melanoma tumor samples and one normal tissue from six patients. This sample set included two metastatic tumors from each patient, and in one instance multiple regions from each metastatic site (7 and 2 regions) in order to assess intratumoral heterogeneity. Importantly, this sample set has associated mRNA expression measured using the nCounter® PanCancer Pathways for Human panel as well as whole exome sequencing (WES) data. Somatic variants seen in this latter dataset were compared with the results from the DNA SNV Solid Tumor Panel. Samples with variants that were detected in the nCounter assay but not WES were subjected to deep sequencing for validation. Overall, we show that this multiplex and multi-omic platform has the potential for rapid and sensitive assessment of patient samples that will impact clinical care. Citation Format: Jinho Lee, Christopher P. Vellano, Gavin Meredith, Jill Mckay-Fleisch, P. Martin Ross, Michael Tetzlaff, Alexandre Reuben, Courtney Hudgens, Jennifer Wargo, Jessica Garber, Andrew White, Joseph Pan, Mike Krouse, Mekala Pansalawatta, Lucas Dennis, Anisha Kharkia, Erin Piazza, Afshin Mashadi-Hossein, Rich Boykin, Nathan Elliott, Brian Filanoski, Gokhan Demirkan, Sarah Warren, Gary Geiss, Dae Kim, Joseph Beechem, Gordon B. Mills. 3D Biology™ view of cancer: Simultaneous detection of somatic DNA mutations and expression profiling of genes and signaling proteins from melanoma tumor FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5563. doi:10.1158/1538-7445.AM2017-5563

Background: Melanoma therapy has benefitted greatly from immune checkpoint blockade, although responses are variable and not always durable. There is a growing appreciation of the role of the microbiome in cancer-related outcomes and recent evidence in murine models suggests that modulation of the gut microbiome may enhance responses to immune checkpoint blockade in melanoma. However this has not been investigated in patients. Here, we demonstrate that differential bacterial “signatures” exist in the gut microbiome of responders (R) and non-responders (NR) to anti-PD-1 therapy, and that insights gained could be used to derive actionable strategies to enhance responses. Methods: We collected buccal (n=105) and stool (n=53) samples from a cohort of anti-PD-1 treated metastatic melanoma patients (n=110). Patients were classified as either R or NR based on RECIST criteria, and 16S rDNA, and whole-genome shotgun sequencing was performed to characterize the diversity, composition and functional capabilities of the microbiomes. Immune profiling (via 7-marker IHC panel of CD3, CD8, PD-1, PD-L1, Granzyme B, RORγT and FoxP3) and cytokine analyses were also performed on available tumors and serum samples at baseline. Results: In these studies, we observed significant differences in the diversity and composition of the gut microbiome in R versus NR to PD-1 blockade at baseline, but no clear differences in buccal microbiomes. Specifically, R had a significantly higher alpha diversity compared to NR (p=0.017), and the Ruminococcaceae family of the Clostridiales order was enriched in R whereas Prevotellaceae family of the Bacteroidales order was enriched in NR. Immune profiling demonstrated significantly increased immune infiltrates in baseline tumor samples of R, with a positive correlation between CD8, CD3, PD-1 and FoxP3 T-cell density and abundance of specific bacteria enriched in R (e.g. Faecalibacterium). Low diversity was also associated with elevated levels of chronic inflammation markers in the serum at baseline. Lastly, we saw differentially abundant metabolic pathways in the gut microbiomes of R (pyrimidine nucleotide biosynthesis, fatty acid biosynthesis, shikimate pathway) vs NR (Tricarboxylic acid cycle, assimilatory sulphate and nitrate reduction, tryptophan biosynthesis). Conclusion: Differences exist in the diversity and composition of the gut microbiome in R vs NR to anti-PD-1 therapy and these microbiota could bridge the gap between host metabolism and anti-tumor immunity. These results have far-reaching implications and suggest that modifications to the gut microbiome could potentially enhance therapeutic responses to immune checkpoint blockade. Citation Format: Vancheswaran Gopalakrishnan, Christine Spencer, Alexandre Reuben, Peter Prieto, Diego Vicente, Tatiana V. Karpinets, Courtney W. Hudgens, Diane S. Hutchinson, Michael Tetzlaff, Alexander Lazar, Michael A. Davies, Jeffrey E. Gershenwald, Robert Jenq, Patrick Hwu, Padmanee Sharma, James Allison, Andrew Futreal, Nadim Ajami, Joseph Petrosino, Carrie Daniel-MacDougall, Jennifer A. Wargo. Response to anti-PD-1 based therapy in metastatic melanoma patients is associated with the diversity and composition of the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2672. doi:10.1158/1538-7445.AM2017-2672

There have been significant advances in the treatment of metastatic melanoma through targeted and immunotherapy, however a significant proportion of patients still progress on these regimens with many experiencing mixed responses. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. The appreciation of genetic heterogeneity as a contributor to resistance to therapy has grown, though immune heterogeneity has been poorly characterized. The goal of the present study is to better understand the molecular and immune heterogeneity in synchronous melanoma metastases at the time of disease progression. In this study, we prospectively evaluated 32 tumors from 15 patients who were treatment-naïve (n = 4), or had received prior targeted (n = 4) or immunotherapy (n = 7). Whole exome sequencing demonstrated between 4-41% of non-synonymous exonic mutations (NSEM) were restricted to individual tumors within a patient. Deep profiling of infiltrating immune cell subsets by flow cytometry and immunohistochemistry analyses confirmed the immune infiltrate between synchronous metastases to be highly heterogeneous, specifically in regards to CD4 and CD8 T lymphocytes. In aggregate, 92% of these T cell clones were unique to distinct tumors within the same patient, with limited overlap with clones detected in the blood. NetMHC3.4 neoantigen prediction demonstrated a large fraction of predicted neoantigens were restricted to individual tumors, with over 10% of these presenting extremely high predicted affinity. Importantly, analysis of RECIST measurements of individual lesions within the same patient suggested this molecular and immune heterogeneity could contribute to differential tumor growth and response to therapy within the same patient. This has important clinical implications, and suggests a single tumor biopsy may not be sufficiently representative of the molecular and immune landscape of multiple tumors within the same individual. Citation Format: Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Xizeng Mao, Wei-Shen Chen, Hannah Cheung, Hong Jiang, Cara Haymaker, Mariana Petaccia De Macedo, Haven R. Garber, Pei-Ling Chen, Vancheswaran Gopalakrishnan, Jacob Austin-Breneman, Courtney W. Hudgens, Jason Roszik, Patrick Hwu, Scott E. Woodman, Lynda Chin, Michael A. Davies, Rodabe N. Amaria, Sapna P. Patel, Alexander J. Lazar, Michael T. Tetzlaff, Karen C. Dwyer, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, Jianhua Zhang, Andrew Futreal, Zachary A. Cooper, Jennifer A. Wargo. Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2392.